The formation and specification of mesoderm results in the generation of a wide range of tissues, including blood, endothelium, heart and skeletal muscle. Many of the known regulatory molecules in these process were identified through the use of model systems such as Xenopus, Zebrafish and the chick embryo that provide access to cell population undergoing mesoderm commitment. The goals of the experiments in this proposal build on preliminary findings which demonstrate that ES cells, engineered to express the green fluorescence protein (GFP) under control of the brachyury locus provide an in vitro model for studying mesoderm induction. With this model, cells expressing the mesoderm gene brachyury can be monitored and/or isolated based on GFP expression. In the first aim of the proposal, we propose to define the kinetics of mesoderm development in this ES model and characterize the full developmental potential of the GFP+ cells. The second aim will focus on understanding the role of known mesoderm regulators on the development and specification of the ES cell derived GFP+/brachyury+ cells. In the third aim, we will isolate mesoderm subpopulations with different developmental fates and define their molecular differences by Affymetrix microarray analysis. The information from these studies will provide a better understanding of the development of mesoderm-derived cell populations and tissues in the early embryo. This information could ultimately provide insights into Underlying causes of disease of the hematopoietic and cardiovascular system.